• Master of Science from the Department of Botany, University of Calcutta, West Bengal
• Ph.D. in Molecular Virology from the Indian Institute of Science, Bangalore, Karnataka
• Post-doctoral researcher in the Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
• Senior Research Associate, Centre of Excellence in Hepatitis C virus research, Indian Institute of Science, Bangalore, Karnataka
• Research Scientist-C, Translational Health Science and Technology Institute, NCR Biotech Science Cluster

Philosophy of research work: Over millions of years of evolution the mammalian immune system has armed itself with many types of weaponry to fight pathogenic microbes. Advantaged by their smaller genome and faster generation time the latter continue to pose immense challenges to the physiological well-being of humans. Although able to successfully ‘respond’ to an infection by most pathogens the human immune system however tends to ‘react’ to that by a few. This leads to death and debility due to hyperinflammatory reaction initiated as a consequence of infection. It is therefore very important to understand the pathobiology of infectious diseases from the host point of view, so that therapeutic strategies can be designed or devised to mellow the ‘reaction’ to a ‘response’, eliminating the consequences of hyperinflammation. 

A very significant part of the research from our group has been performed in collaboration with institutional and extra-institutional scientist. For over a decade now we have been working on pathobiology of Flaviviruses, a group which includes multiple human pathogens like Japanese encephalitis virus (JEV) and Dengue virus (DENV). Initially, we were interested in studying the role played by infection-induced changes in cellular proteostasis that manifests as Endoplasmic Reticulum (ER) stress. Subsequently, we initiated research into understanding the cellular and molecular basis of DENV-infection induced thrombocytopenia or acute drop in platelet level. Additionally, we are interested in establishment of an anti-viral drug discovery platform for identification of ‘hit’ molecules. Subsequent to the global pandemic caused by SARS-CoV-2 infection, we have diverted our efforts to understanding the inflammatory response launched as a response to its infection. We have interesting leads from this research which we hope would lead to design of novel therapeutic regime for COVID19. 

A.    Relation between ER-stress and outcome of virus infection

Introduction: 

In response to infection by a virus our immune system launches its innate and adaptive anti-viral arsenal with the objective of limiting virus replication and clearing infectious virus or virus-infected cells. The Endoplasmic reticulum (ER) is intimately associated with these processes at multiple levels. A significant proportion of mammalian proteins, which are either secreted or destined for the plasma membrane, are folded and glycosylated inside the ER-lumen. This compartments harbours a battery of proteins (foldase and chaperones) that catalyse these steps in protein maturation. An increase in the load of proteins that need to be thus processed leads to an accumulation of unfolded/misfolded proteins in the lumen, a situation called as the ER-stress. This serves as a signal for the ER to initiate a series of biochemical steps called as Integrated-stress response (ISR) or Unfolded-protein response (UPR).  The UPR involves steps that 

a)     reduce the flux of protein into the ER-lumen (inhibition of protein synthesis)

b)     extrude or flush out misfolded proteins from the ER-lumen into the cytosol for degradation

c)     increase the protein folding capacity of the ER-lumen

d)     determine cell survival 

In context of an infection the ER in different cell types play crucial roles in different aspects of the host response which includes

a)     determining the survival of an infected host cell

b)     regulation of the innate antiviral response pathways

c)     regulating the maturation and polarization of macrophages which influences the inflammatory response

d)     production of secretory antibody from plasma cells

Since the consequences of an UPR is relevant to the outcome of multiple metabolic diseases, a number of drugs targeting different signalling axes in this pathway known and more are being developed. Therefore, the study of infection-induced UPR can open new windows of therapeutic strategy involving repurposing of such drug(s) that can help in either curing virus infection or managing the consequences of virus infections better. 

Accomplishment:

An accumulation of unfolded proteins in the ER-lumen activates three ER-membrane trans-membrane proteins, IRE1a, PERK and ATF6 that initiate the UPR. As an immediate effect of PERK and IRE1a activation, translation of ER-bound mRNAs is impeded through post-translational modification of initiation factors (eIF2a) by PERK and direct cleavage of mRNAs by IRE1a(RIDD pathway). In view of the fact that UPR activation in an infected host cell, can potentially suppress virus replication through an effect on viral protein synthesis, we explored the effect of pharmacologically suppressing these sensors. Surprisingly, inhibition of PERK activation did not affect virus replication but that of IRE1a showed an unexpected effect. In contrast to our expectation, we observed a drop in virus production upon inhibition of ribonuclease domain of IRE1a through an unknown mechanism (Bhattacharyya et al, Journal of General Virology, 2014). The potential mechanisms include the inhibition of this nuclease to have a negative effect on one or more pro-viral factor(s), which are expressed or activated as part of the UPR or viral molecular mimicry of those host proteins, the mRNA of which are not targeted by this nuclease (discussed in Frontiers in Microbiology, 2014).  The third sensor involved in UPR, i.e. ATF6 showed its involvement in Autophagy pathway that is also activated in response to infection . In collaboration with the lab of Dr Manjula Kalia (RCB) we observed UPR to be a trigger for subsequence activation of Autophagy in viral infected cells, which was observed to have a negative effect on viral life cycle through its influence on host innate antiviral pathways (Autophagy, 2014; Journal of General Virology, 2017). Long non-coding RNAs, once considered physiologically inconsequential, are increasingly being proven to play crucial role in different cellular pathways including anti-viral response. Comparison of the transcriptome between cells are either uninfected or infected with multiple Flaviviruses showed a moderate but significant deregulation of the pleiotropic lncRNA Malat1 (Scientific Reports, 2015). A deeper characterization of the responsible signalling pathway implicated activation of the PERK sensor, an ER-membrane resident sensor of unfolded proteins in the ER-lumen, to directly regulate Malat1 expression (Scientific Reports, 2015). In addition to Malat1 a large number of lncRNA, most of them functionally uncharacterized, showed deregulation in virus infected cells with unknown consequences (our unpublished data). The IRE1a catalysed RIDD-pathway is known to cleave host mRNAs and select microRNA precursors. Interestingly, we observed negative regulation of a few lncRNA by IRE1a through activity of the ribonuclease domain of this protein (our unpublished data). The implications of such regulation for virus induced UPR in particular and ER-stress pathway in general would require extensive characterization.   

B.    Studies in thrombocytopenia in relation to Dengue virus

A characteristic clinical feature of Dengue virus infection is an acute drop in blood platelet level or thrombocytopenia, an outcome implicated in infection associated haemorrhage. Multiple  mechanisms have been proposed by different research groups as the aetiology of this thrombocytopenia, each probably contributing partially. These include: 

a)     an inhibition of platelet production following infection of platelet mother cells called Megakaryocytes and 

b)     enhancement of degradation following generation of anti-platelet antibody through molecular mimicry and/or direct infection of platelets. 

We use different model systems to study the effect of virus infection on platelet production and stability. The human K562 cell line represents a bipotential Megakaryocyte-Erythrocyte progenitor (MEP) stem cell, that can be differentiation into cells representing either lineage by the use of differential pharmacological stimuli. When stimulated to generate Megakaryocytes these cells expand, undergo endomitosis and express platelet-specific surface markers, akin to bone marrow resident version of these cells. Financially supported by an extramural research grant from the SERB, Department of Science and Technology, Govt. of India, we observed that in differentiating cells infected with Dengue virus at least two crucial differentiation steps, expression of surface markers and endomitosis, are suppressed. Comparison of the signalling axes stimulated during differentiation implicated interference with specific MAP-Kinase pathway in infected cells. Differentiation into Megakaryocytes involve unique changes in the transcriptional landscape. We have performed detailed transcriptome analysis of differentiating cells that are either uninfected or infected cells at various time points. Currently we are analysing the data to look into differentiation-associated gene expression pattern in uninfected cells that are disturbed following infection. We would like to apply the data of such differential expression in the Connectivity map database, in order to predict known pharmaceuticals that can reverse the changes caused by infection (BIORXIV/2020/172544). As an interesting observation, we saw a promotion of virus replication in cells that are differentiating into megakaryocytes when compared to either undifferentiated cells or cells differentiating into erythrocyte lineage. Preliminary investigation into the mechanism of this promotion in viral replication indicated enhanced translation of viral genomic RNA leading to higher viral replication. This probably adds credence to the hypothesis that bone marrow megakaryocytes serve as an early reservoir of Dengue virus replication (BIORXIV/2020/172544).

In close collaboration with the lab of Dr Prasenjit Guchhait in the Regional Centre for Biotechnology, we have shown that in vitro Dengue virus can interact with platelets from healthy human peripheral blood and activate them (Ojha et al, 2017). Although the molecular details of this activation are still not very clear, it was observed that platelets thus activated seem to release soluble factors which then promote or augment the replication of Dengue virus in already infected primary monocyte. Aided by an extramural research grant from the Department of Biotechnology, Govt. of India to Dr. Guchhait (as PI), Dr Naval Vikram (AIIMS, co-PI) and me (co-PI), further characterization of this observation lead to identification of CXCL4, a cytokine released from activated platelets to interact with its cognate receptor CXCR3 on infected monocytes and promote viral replication in these cells. As a proof of this being a physiologically relevant, the level of this cytokine in severe Dengue patients was observed to be very high. Additionally, AMG487 a drug known to inhibit signalling by CXCR3 prevented the replication promotion caused by CXCL4 (Ojha et al, 2019). At present, our collaborative effort is directed towards discovery of similar novel inhibitors of CXCR3 that can show the same function. In this we are supported by a research grant to Dr. Guchhait (as PI), Dr. Shailendra Asthana (THSTI, as co-PI) and me (co-PI) from the SERB, Department of Science and Technology, Govt. of India. Our goal is to use structure-based computer-aided drug discovery (CADD) protocols for rational design of small molecule, which can then be tested for functional activity. 

C.    Directly acting antiviral for inhibition of dengue virus replication 

The combat against viral diseases uses either of the two principal arsenal of prophylactic vaccination or therapeutic intervention. Development of an universally applicable or acceptable vaccine to provide protection from Dengue virus is complicated by the development of severe disease by presence of sub-neutralizing antibodies. We in collaboration with Dr. Shailendra Asthana (THSTI, Computational biophysicist) and Dr. Rambabu Gundla (GITAM University, Synthetic chemist) have initiated efforts towards rational design of small molecule inhibitor targeting the viral RNA-dependent RNA polymerase enzyme, which is responsible for replication of the viral genome. Inhibition of similar enzyme has proven very efficacious in preventing death and debility for patients infected with at least two viruses, for which there are not vaccines available till date, namely Hepatitis C virus and Human Immunodeficiency virus. Designed using structure-based CADD protocols, we have discovered two novel molecules that are predicted to bind to allosteric site on the Dengue virus RdRp and inhibit viral replication. The molecules have shown efficacy in ex vivo model using cultured cell line, with high Selectivity Index (SI). The Structure-Activity relation (SAR) study of these molecules have been performed and a PCT application filed (PCT application number:PCT/IN2021/050596). A manuscript providing greater details regarding the activity of these molecules is currently being prepared. 

D.    Studies in COVID-19

The pandemic caused by SARS-CoV-2 infection is largely due to the novel nature of the infecting virus making it refractory to adaptive memory immune response induced following infection by predecessor benign cousins of the virus. We have diverted our lab resources to two principal directions in order to aid and abet efforts to counter the pandemic. On the one hand we have standardized in vitro virus neutralization assays, to be performed in biosafety level-3 laboratory using live virus, to qualitatively and quantitatively assess neutralizing antibodies generated following vaccination or that in therapeutic antibodies. Our efforts have helped many corporates to evaluate the efficacy of their vaccine candidates, notable among which include Dr Reddy’s laboratory to develop the Sputnik-V vaccine and Mynvax to develop an indigenous sub-unit vaccine. In addition to that we have collaborated with academic organizations to support sero-surveillance programs, which in addition to institutional programs includes IISER-Pune, TIFR-Mumbai, AIIMS-New Delhi and CSIR-IGIB, New Delhi. 

Beside this, our group has collaborated with the research group of Dr. P. Guchhait in RCB for studying aspects of the inflammatory response induced by SARS-CoV-2 infection. Based on studies of the basic biology of infection in a hamster model, we have shown the efficacy of a nutritional supplement in attenuating the lung damage caused by virus infection. The result of this study has been submitted for publication and is currently under review. In another study in collaboration with Dr Guchhait and Dr Suman Das in ESIC hospital, Faridabad, we have studied platelets isolated to COVID19 patients to understand the molecular basis of the observed hypercoagulability. The result of this work is also currently under review.